Lab Protocols

To take a multichannel image in Elements: 1. click acquire --> capture multi channel image --> manually 2. There should now be an open window with tabs along the bottom for each of the image channels 3. Bright-field should be included in those tabs 4. If Bright-field is not included, you will need to add a channel. a. click acquire --> capture multichannel image --> multichannel setup b. click on an unchecked black box to add a channel, assign the channel the appropriate settings c. for Bright-field set the comp. color to Bright-field d. when you close re-open the multichannel image you should have a bright-field tab 5. Click the tab along the bottom that corresponds with the image you want to take 6. set-up the image using the microscope or i-control 7. click capture when you are ready to take the picture 8. Take all of the images you need to compile and then click the four box icon in the left hand corner. The icon is a shaded box with three colored boxes p...

This protocol explains how to: use Olympus fluorescent microscopes to image in vitro experiment slides.   Protocol written by: Julie Ruble Turn on green burner power button first, and then the microscope and then the camera. Start viewing in fluorescence (change filters using the filter wheel until you reach the green filter). Begin at the corner of the slide. Use the 40X objective lens. When looking for neurons to image, scan through the slide methodically so as not to miss any: When you find a neuron, position it with the cell body in the field of vision but off to the side (so you'll be able to get more neurite in the picture and therefore take fewer pictures). NOTE: When you are not looking at or imaging the neuron, close the shutter on the right side of the microscope so as not to bleach the neuron. To take a picture, open SPOT (the program may have a shortcut on the desktop, but you may need to find it in the C folder and create one). Then, to take a flu...

How to clean an objective:  Turn off the microscope. Turn the nosepiece until the objective in question is at an angle proper for removal or cleaning (See Figure 1).     Figure 1. If you choose to remove the objective for cleaning, very carefully unscrew it from the nosepiece and gently place it on a Kim-wipe on a clean, flat surface. Whether the objective is still attached to the nosepiece or not, the next step is to cover a Q-tip in lens paper and soak the tip in either alcohol or lens cleaning solution. (Lens-cleaning solution can be found in a small, zippered bag on the shelf for the Olympus microscope in Dana) (See Figures 2 and 3). Figure 2.   Figure 3 . Gently rub the glass on the objective—the part that is adjacent to the sample when viewing- with the wet Q-tip. Do NOT touch the inside of the objective where it attaches to the microscope. When you feel that you have sufficiently cleaned the dirt or oil from the objective, acquire...

This protocol explains how to: add a scalebar to an image. Protocol written by: Barbara Lom 1. To include a scale bar on any image you need to acquire the image and an image of the micrometer (tiny ruler) slide at the same magnification and image parameters. 2. Open your "ruler" image in Photoshop. If the image is a bit askew you will need to rotate the image so that the ruler marks are "square" in the frame of your picture (it is OK to crop the ruler image if necessary in this step). 3. Go to the "layers" box (usually at the lower right - if not then make layers visible under the "window" menu & select "show layers") 4. Click on the arrow in the circle in the upper right corner of the layers box and select "new layer" 5. Go to the toolbar and select the pencil tool (which is sometimes hiding under the paintbrush tool). 6. Click on brush arrow (above the image) to change the "width" ...

This protocol explains how to: align the phase on the Olympus fluorescent microscope.   Protocol written by: Barbara Lom, Fiona Watson, Julie Ruble NOTE: While following this protocol, please reference the labeled diagram found below.  A full-sized version of this image is available here . Start with the 10X objective and make sure the Phase Selector Ring is on "Ph 1."  Focus on a slide. Take out the right eyepiece of the Olympus fluorescent microscope and insert the UT30 centering telescope (found bubble-wrapped in a plastic jar by one of the scopes). Push in and turn allen wrench knobs until both rings viewed through the telescope eyepiece align in the dark circle.  Make sure the allen wrenches are catching in the screw hole when you push them in to turn them. Repeat this with each objective and its corresponding Phase Selector Ring setting.  For the 40X objective, the ring should be on "Ph 2," and for a 100X objective, it should be set on "Ph 3....

This protocol explains how to: take pictures with the Nikon inverted scopes using ImagePro.   Protocol written by: Barbara Lom Turn on the power to the microscope and check that the light is directed to the eyepieces by setting the dial on the upper right hand side to the “bino” setting. Use the 10X objective to find and focus on your sample.  Adjust the light intensity is adjusted with the dial on the lower/front/left of the microscope.  If you are using phase contrast optics make sure the phase ring is in place (slider near the top of the microscope). Adjust the focus and lighting to get your best phase contrast image. Divert light from oculars to the camera by turning the dial from “bino” to “photo” setting. Turn the camera power on (small beige box). Turn on the PC and log into Windows. Open ImagePro+ (Microscope Icon – upper left side of desktop). Select the “complete” option and click “OK”.  Ignore the registration request and close the mac...

This protocol explains how to: Image Using a 100x Objective (Oil Immersion) Protocol revised: 11/17/10 Protocol written by: Aaron Deal Note: When imaging a slide using a 100x objective, oil is required for a precise image. 1. View the slide on a compound microscope. Begin on a lower powered objective and then move up to a dry 100x objective in order to find the desired, or potentially desired, image. (If the image is brightfield, see the Kohler illumination protocol for instructions on setting up Kohler illumination) 2. Once the image is in view, rotate the nosepiece so that no objective is pointing towards the slide. 3. Place 1-2 drops of objective lens oil (located on the shelves above the microscope) directly onto the slide where the objective will be located. 4. Rotate the nosepiece back to the 100x objective, without passing any other objectives through the oil (the 100x objective lens should now be surrounded by the oil). 5. The stage can still be moved, but a reappli...

Use this protocol if you need to prepare a specimen for imaging with a microscope that has a small working distance (such as the confocal) and preserve it for storage afterward. 1) Gather the supplies you'll need: (also listed below this image) Microscope slides - preferably from a box of plain, precleaned, slides with the dimensions 25x75x1mm Plastic pipette (or glass, if you prefer) Sharpie or another permanent marker Nail polish Forceps (with black tape on the handle, which designates use with fixed specimens only) Reinforcement labels Slide covers (coverslips) - preferably from a box that lists the dimensions as "22x22-1.5um" Fluoro-gel 2) Obtain your prepared (fixed, sliced) samp le. 3) On a slide, label your initials, experiment, and date with your permanent marker: 4) Place one or two reinforcement labels on the slide. If you choose only one, place it in the center of the slide; if two, place them evenly spaced out. The latter is usually preferred...

1) Open Photoshop on the computer 2) Click File > Open 3) Select the images that you want to overlay and open them in photoshop 4) Make sure there is a saved copy of all the original images 5) Save the images under new names before you begin manipulating them, so that you will always have an unchanged original 6) Once you have opened all of your images, pick one to be your base image (In this example, the base image will be green, and the other two images will be red and blue). 7) Bring the blue fluoresent image to the front 8) Go to Image> Select> All 9) Then, go to File> Copy (or Ctrl C) 10) Once you have a copy of the blue image, go back to your base green image and click on the channels tab (next to the layers tab at the bottom right) 11) Highlight the blue channel 12) Paste (File> Paste or Ctrl V) your copied blue image into the blue channel 13) Repeat these steps copying the red image into the red channel 14) Now save the overlay image as a photoshop a...

1) Open Photoshop on the computer 2) Click File > Open 3) Select the images that you want to overlay and open them in photoshop 4) Make sure there is a saved copy of all the original images 5) Save the images under new names before you begin manipulating them, so that you will always have an unchanged original 6) Once you have opened all of your images, decide how many panels you need for your final image, and pick one to be your base image 7) On your base image, go to Image> Canvas Size 8) A new box will open with the height and width of your current canvas 9) Multiply the height or width by the number of images that need to fit within your canvas (This means if you want a three panel image multiply the width by three and do not change the height. If you want a four panel square image, multiply the width and height by two). 10) Once you change the canvas height and width to the desired size, anchor the base image where you want it in the final image panel (Ex. If you ar...

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments. A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order, obtaining...